Our studies in FY2015 have addressed several major questions: 1. The major goals of this study were to perform an in depth characterization of the gene signature of human T regulatory cells (Tregs). Highly purified Treg and T conventional (Tconv) subsets either freshly explanted or activated in vitro were analyzed by transcriptome sequencing (RNA-seq) to completely profile mRNA expression. Gene expression was validated using the nCounter system. In addition to RNA-seq, we analyzed microRNA (miRNA) expression using microarray technology. We confirmed previous studies demonstrating that certain genes are differentially expressed in Tregs including, but not limited to, FoxP3, IKZF2, and CTLA4. A number of novel genes (RTKN2, LAYN, UTS2, CSF2RB, TRIB1, F5, CECAM4, CD70, ENC1 and NKG7) were identified and validated to be DE in Tregs and Tconv sorted from multiple healthy donors. We attempted to characterize the potential roles of RTKN2 and LAYN in Treg function by using overexpression or siRNA knockdown followed by in vitro human Treg suppression assays. We were able to perform a predictive analysis to generate an interactome based on DE genes and miRNAs in Tregs and Tconv. These potential interactions between miRNAs and mRNA selectively expressed in Treg should facilitate the development of approaches to validate the functional significance of these predictions. 2. We have developed a CRADA with the Department of Biotherapeutics at Boehringer-Ingelheim (BI) Pharmaceuticals for studies to generate novel mAbs that will modulate the function of human Treg cells. We have prepared large numbers of expanded highly purified human Tregs that are used to immunize mice to generate the mAbs. A number of novel screening techniques have been developed by BI to identify mAbs that selectively recognize human Tregs and not activated T conventional cells or non-lymphoid cells. These novel mAbs are then tested in a panel of in vitro T suppressor cell assays that we have developed. In FY2015, we screened hundreds of supernatants and identified supernatants that selectively react with human Tregs and/or modulate the function of Tregs when added to in vitro assays. Several of these antibodies havwe been purified from supernatants. Purified antibodies will be used in immunoprecipitation assays to identify the target antigen by mass spectroscopy. 3. Tregs have a diminished capacity to activate the PI3K/Akt pathway. Although blunted Akt activity is necessary to maintain Treg function, the consequences of this altered signaling are unclear. Glucose transporter 1 (Glut1) is a cell surface receptor responsible for facilitating glucose transport across plasma membranes whose expression is tightly coupled to co-stimulatory signals and Akt phosphorylation. Freshly isolated human Tregs were unable to up-regulate Glut1 in response to TCR and co-stimulatory signals as compared to conventional T cells. Consequently, the ability of Tregs to utilize glucose was also reduced. Introduction of Foxp3 into conventional T cells inhibited both Akt activation and Glut1 expression, indicating that Foxp3 can regulate Glut1. Finally, pharmacologic activation of Akt in Tregs can induce Glut1, overcoming the effects of Foxp3. Together, these results illustrate the molecular basis behind differential glucose metabolism in Tregs.